DNA-sorb-B. Nucleic acid Extraction kit Instruction Manual

Transcription

1 For Professional Use Only DNA-sorb-B Nucleic acid Extraction kit Instruction Manual AmpliSens Ecoli s.r.o., Studenohorska Bratislava 47 Slovak Republic Tel.: Fax: Federal Budget Institute of Science Central Research Institute for Epidemiology 3A Novogireevskaya Street Moscow Russia

2 TABLE OF CONTENTS 1. INTENDED USE PRINCIPLE OF NUCLEIC ACID EXTRACTION CONTENT ADDITIONAL REQUIRMENTS GENERAL PRECAUTIONS SAMPLING AND HANDLING WORKING CONDITIONS PROTOCOL TROUBLESHOOTING TRANSPORTATION STABILITY AND STORAGE REFERENCES QUALITY CONTROL KEY TO SYMBOLS USED... 9 REF K CE; REF K CE / VER / Page 2 of 10

3 1. INTENDED USE DNA-sorb-B nucleic acid extraction kit is intended for extraction and purification of DNA from the clinical material (whole blood, plasma, urine sediment, saliva, cerebrospinal fluid, sputum, biopsy material, bronchoalveolar lavage, feces). 2. PRINCIPLE OF NUCLEIC ACID EXTRACTION DNA-sorb-B nucleic acid extraction kit is a reagents kit for rapid and efficient manual extraction and purification of DNA from various clinical material. Lysis solution contains chaotropic agent (guanidine thiocyanate) that lyses cells and denaturates cell proteins. The nucleic acids are then sorbed on silica particles. DNA extracted from clinical samples may be used for PCR diagnostic tests. 3. CONTENT DNA-sorb-B nucleic acid extraction kit is produced in 2 forms: DNA-sorb-B nucleic acid extraction kit variant 50, REF K CE; DNA-sorb-B nucleic acid extraction kit variant 100, REF K CE; DNA-sorb-B nucleic acid extraction kit variant 50 or 100 includes: Reagent Lysis Solution Description clear liquid from colorless to yellow or pink colour Variant 50 Variant 100 Volume, ml Quantity Volume, ml Quantity 15 1 vial 30 1 vial Washing Solution 1 colorless clear liquid 15 1 vial 30 1 vial Washing Solution 2 colorless clear liquid 50 1 vial vial Universal Sorbent TE-buffer for DNA elution suspension from white to dark beige colour tube tubes colorless clear liquid tube tubes DNA-sorb-B nucleic acid extraction kit variant 50 is intended for 50 reactions, including controls. DNA-sorb-B nucleic acid extraction kit variant 100 is intended for 100 reactions, including controls. 4. ADDITIONAL REQUIRMENTS Vacuum aspirator with flask for removing supernatant. Disposable powder-free gloves and a laboratory coat. Pipettes (adjustable). REF K CE; REF K CE / VER / Page 3 of 10

4 Sterile pipette tips with aerosol filters (up to 200 µl and 1000 µl). Vortex mixer. Desktop microcentrifuge with rotor for 2 ml reaction tubes (RPM max. 16,000). PCR box or Biological cabinet. Thermostat for tubes with controlled temperature for C. Tube racks. Disposable 1.5-ml polypropylene tubes. Refrigerator for 2 8 C. Deep-freezer at the temperature from minus 24 to minus 16 C. Reservoir for used tips. 5. GENERAL PRECAUTIONS The user should always pay attention to the following: Use sterile pipette tips with aerosol filters and use a new tip for every procedure. Store all extracted positive material (specimens, controls and amplicons) away from all other reagents and add it to the reaction mix in a distantly separated facility. Thaw all components thoroughly at room temperature before starting an assay. When thawed, mix the components and centrifuge briefly. Use disposable protective gloves and laboratory cloths, and protect eyes while samples and reagents handling. Thoroughly wash hands afterwards. Do not eat, drink, smoke, apply cosmetics, or handle contact lenses in laboratory work areas. Do not use a kit after its expiration date. Dispose of all specimens and unused reagents in accordance with local regulations. Samples should be considered potentially infectious and handled in biological cabinet in compliance with appropriate biosafety practices. Clean and disinfect all samples or reagents spills using a disinfectant, such as 0.5 % sodium hypochlorite or another suitable disinfectant. Avoid samples and reagents contact with the skin, eyes, and mucous membranes. If these solutions come into contact, rinse the injured area immediately with water and seek medical advice immediately. Safety Data Sheets (SDS) are available on request. The kit is intended for single use for analysis of specified number of samples (see the section Content ). The kit is ready for use in accordance with the Instruction Manual. Use the kit strictly for REF K CE; REF K CE / VER / Page 4 of 10

5 intended purpose. Use of this product should be limited to personnel trained in DNA amplification techniques. Workflow in the laboratory must be one-directional, beginning in the Extraction Area and moving to the Amplification and Detection Area. Do not return samples, equipment and reagents in the area where the previous step was performed. Contains substance: guanidine thiocyanate. Lysis Solution, Washing Solution 1 H302: Harmful if swallowed. H312: Harmful in contact with skin. H314: Causes severe skin burns and eye damage. H332: Harmful if inhaled. H412: Harmful to aquatic life with long lasting effects. EUH032: Contact with acids liberates very toxic gas. Danger Washing Solution 2 Warning Universal Sorbent Warning P260: Do not breathe vapours. P264: Wash your hands thoroughly after handling. P273: Avoid release to the environment. Р302+Р352: IF ON SKIN: Wash with plenty of water P501: Dispose of contents in accordance with national regulation. Contains substance: isopropyl alcohol H226: Flammable liquid and vapour. H319: Causes serious eye irritation. H336: May cause drowsiness or dizziness P210: Keep away from heat, hot surfaces, sparks, open flames and other ignition sources. No smoking. P261: Avoid breathing vapours. P264: Wash your hand thoroughly after handling. P305+P351+P338: IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses if present and easy to do continue rinsing. P403+P233: Store in a well ventilated place. Keep container tightly closed. P501: Dispose of contents in accordance with national regulation. Contains substance: Celite H373: May cause damage to organs through prolonged or repeated exposure P260: Do not breathe dust/fume/gas/mist/vapours/spray. Р314: Get medical advice if you feel unwell. P501: Dispose of contents in accordance with national regulation. REF K CE; REF K CE / VER / Page 5 of 10

6 6. SAMPLING AND HANDLING Obtaining of clinical material samples for PCR-analysis, transportation and storage are described in manufacture s handbook [2]. It is recommended to read this handbook before starting of the work. DNA-sorb-B nucleic acid extraction kit is recommended for DNA extraction and purification from: whole blood, blood plasma, urine sediment, saliva, cerebrospinal fluid, sputum, biopsy material, bronchoalveolar lavage, feces. 7. WORKING CONDITIONS DNA-sorb-B nucleic acid extraction kit should be used at C. 8. PROTOCOL 8.1. DNA Extraction Volume of clinical sample for DNA extraction is 0.1 ml. 1. Warm Lysis Solution and Washing Solution 1 (if stored at 2-8 ºС) at the temperature 65 ºС until the crystals disappear. 2. Take the required number of 1.5 ml disposable tubes including one tube for Negative Control of Extraction (Negative Control, C-) and one tube for Positive Control of Extraction (Positive Control, PCE) (if provided with the amplification kit)). 3. Add 10 µl of Internal Control (if it is provided for analysis of this infectious agent) and 300 µl of Lysis Solution to each tube. Mark the tubes. 4. Add 100 µl of a sample to the tube using tips with aerosol filter. Add 100 µl of Negative Control (provided with the amplification kit) to the tube labeled C-. Add 90 µl of Negative Control (provided with the amplification kit) and 10 µl of Positive Control (if it is provided for analysis) to the tube labeled PCE. 5. Thoroughly vortex the tubes then incubate at 65 ºC for 5 min (do not heat the tubes if extracting DNA from blood plasma). 6. Centrifuge all tubes at 5,000 rpm for 5 s. If a sample hasn t dissolved completely, centrifuge the tube at 12,000 rpm for 5 min, transfer the supernatant to a clean tube and use for DNA extraction. 7. Thoroughly resuspend Universal Sorbent on vortex mixer. Into each test tube add 25 µl of Universal Sorbent. Carefully vortex the tubes then leave them in a rack for 2 min. Vortex once again and incubate the tubes for 5 min in a rack. 8. Centrifuge all the tubes at 5,000 rpm for 30 s and carefully remove supernatant without disturbing the pellet using a vacuum aspirator. Use a new tip for each tube. 9. Add 300 µl of Washing Solution 1 to each tube. Vortex thoroughly until sorbent is fully REF K CE; REF K CE / VER / Page 6 of 10

7 resuspended. Centrifuge at 5,000 rpm for 30 s. Carefully remove supernatant from each tube without disturbing the pellet using vacuum aspirator. Use a new tip for each tube. 10. Add 500 µl of Washing Solution 2 to each tube. Vortex thoroughly until sorbent is fully resuspended. Centrifuge at 10,000 rpm for 30 s. Carefully remove supernatant from each tube using a vacuum aspirator. Use a new tip for every tube. 11. Repeat step 11. Remove supernatant completely. 12. Incubate the tubes with open caps at 65 C for 5-10 min (to dry the pellet). 13. Add 50 µl of TE-buffer for DNA elution. Vortex the tubes. Incubate the tubes at 65 ºC for 5 min; vortex occasionally while incubating. 14. Centrifuge tubes at 12,000 rpm for 1 min. The supernatant contains purified DNA. The samples are ready for PCR amplification. Be careful not to collect sorbent while removing of the DNA-containing solution. If solution is muddy, centrifuge the tube to precipitate the sorbent. The purified DNA can be stored: at 2 8 C for 1 week; at the temperature not more than minus 16 C for 1 year Amplification It is recommended to use AmpliSens PCR kits. Please carry out the amplification according to the manufacturer instructions. 9. TROUBLESHOOTING These troubleshooting guides may be helpful in explaining any problem that may arise. False negatives with extraction product: Degradation of the nucleic acid contained in the sample. It is necessary to use a new sample. Store samples under appropriate conditions. Loss of nucleic acid pellet. Carefully discard the washing solution trying not to disturb the nucleic acid pellet. Degradation of the extracted nucleic acid. It is necessary to use DNase- and RNasefree plastic. False positives with extraction product: Contamination during sample extraction. It is necessary to open one test tube at a time. Avoid spilling the contents of the test tube, always change tips. Contamination of the reagents prepared for the step. It is necessary to repeat the test. REF K CE; REF K CE / VER / Page 7 of 10

8 Contamination of the extraction zone by amplicons.. It is necessary to clean surfaces and instruments using aqueous detergents, wash lab coats, and replace test tubes and tips in use. Use different laboratory coats in different areas. If you have any further questions or encounter problems, please contact our Authorized Representative in the European Community. 10. TRANSPORTATION DNA-sorb-B nucleic acid extraction kit should be transported at STABILITY AND STORAGE All components of the DNA-sorb-B nucleic acid extraction kit are to be stored at 2-25 ºC, when not in use. They also must be stable until the expiry date stated on the label. The shelf life of reagents before and after the first use is the same, unless otherwise stated. 12. REFERENCES 1. R Boom, C J Sol, M M Salimans, C L Jansen, P M Wertheim-van Dillen and J van der Noordaa; "Rapid and simple method for purification of nucleic acids. "J. Clin. Microbiol. March 1990 vol. 28 no Handbook Sampling, Transportation, and Storage of Clinical Material for PCR Diagnostics, developed by Federal Budget Institute of Science Central Research Institute for Epidemiology of Federal Service for Surveillance on Consumers Rights Protection and Human Well-Being, Moscow, QUALITY CONTROL In compliance with Federal Budget Institute of Science Central Research Institute for Epidemiology ISO Certified Quality Management System, each lot of the DNAsorb-B nucleic acid extraction kit has been tested against predetermined specifications to ensure consistent product quality. REF K CE; REF K CE / VER / Page 8 of 10

9 14. KEY TO SYMBOLS USED Catalogue number Caution Batch code Sufficient for In vitro diagnostic medical device Expiration Date Version Consult instructions for use Temperature limitation IC Internal control Manufacturer GHS07: Exclamation mark Date of manufacture Authorised representative in the European Community GHS02: Flame GHS05: Corrosion GHS08: Health hazard

10 VER KM VV PM PM EM List of Changes Made in the Instruction Manual Location of changes Cover page Content Stability and Storage Key to Symbols Used Cover page, text 5. General precautions, 14. Key to symbols used Through the text 5. General precautions, 14. Key to symbols used Content Essence of changes The phrase For Professional Use Only was added New sections Working Conditions and Transportation were added The Explanation of Symbols section was renamed to Key to Symbols Used The information about the shelf life of open reagents was added The explanation of symbols was corrected The name of Institute was changed to Federal Budget Institute of Science Central Research Institute for Epidemiology Information about hazards was corrected Correction according to the template Information about hazards was rewritten according to the Regulation 1272/2008/ЕС. The colour of the reagents was specified REF K CE; REF K CE / VER / Page 10 of 10